The pathogenicity of Candida spp. is due to enzyme production,
tissue invasion, and their capacity to adhere to oral mucosa. (1) The
adherence of yeast to oral epithelial cells is influenced by
yeast-related factors such as the expression of adhesion proteins, the
presence of germinative tubes, and the production of extracellular
polymers and enzymes. Host-related factors such as sexual hormones, the
presence of fibrin and fibrinogen, (2) and salivary compounds including
mucine, (3) salivary proteins, (4) and secretory IgA (5) may also
influence this process. The effects of these components on the adherence
of C. albicans differ, as some of them increase the adhesion capacity
whereas others show inhibitory activity. (6)

Biasoli et al. (7) observed a correlation between the capacity for
yeast to adhere and its ability to colonize mucosal surfaces. C.
albicans exhibits the highest values of adherence to oral epithelial
cells relative to other Candida species. Bosh et al. (8) verified that
moderate stress may affect the process of microbial colonization and the
adherence of C. albicans to epithelial cells by altering the secretory
activity of salivary glands.

Because adherence is an important virulence factor in Candida, the
inhibition of this process is an important strategy in the prevention of
oral candidosis. C. albicans antigens, host proteins, antifungal agents,
and antibodies have been used to inhibit C. albicans adherence to host
cells. (5) IgA seems to play an important role by causing fungal
aggregation and preventing the adherence to mucosa or oral surfaces. (9)

Adhesion and colonization of the oral cavity by C. albicans is an
initial step in candidosis. The presence of orthodontic and other oral
appliances may alter the oral ecological environment. Hence, these
appliances may tip the balance to favor the existence of Candida
species. (10)

To gain a better understanding about Candida carriage status and
the specific response against this microorganism in orthodontic
patients, we compared the presence of Candida species in saliva, their
adherence to oral epithelial cells, and the levels of anti-C. albicans
IgA in children with or without orthodontic appliances.

Methodology

The present study was submitted to and approved by the Sao Jose dos
Campos/UNESP Dental School Ethics Committee (process number
078/2002--PH/ CEP).

The study included 30 children 5 to 12 years old (9.1 [+ or -] 1.7
years old) who were users of removable orthodontic appliances for at
least 6 months (experimental group; EG) and 30 children of the same age
(7.7 [+ or -] 1.5 years old) who were not users of any orthodontic
appliances (control group; CG).

Sampling

Intra-oral examination was performed to evaluate the presence of
lesions suggestive of candidosis. Oral mucosa was collected using a
wooden spatula from the cheek and the lateral surface of the tongue of
each patient.

Saliva samples (2 mL) were also collected in sterile tubes and
transported on ice to the microbiology laboratory to be assayed within 3
hours. The saliva samples were diluted 1:10 and 1:100 with sterile
saline and immediately assayed for Candida isolation and identification.
A fraction of 1 mL of each sample was also transferred to sterile tubes
containing 5.0 mM phenylmethylsulfonyl fluoride and 0.002% sodium azide,
and stored at -20[degrees]C until antibody analysis.

Candida isolation and identification

The undiluted and diluted saliva samples (0.1 mL) were plated in
duplicate in Sabouraud dextrose agar (Difco, Detroit, USA) supplemented
with 0.1 mg/mL chloramphenicol (vixmicin, Uniao Quimica, Sao Paulo,
Brazil). After incubation at 37[degrees]C for 48 h, the colonies were
counted and the number of colony-forming units per milliliter (CFU/ mL)
was calculated. Saliva samples were also plated on CHROMagar (CHROMagar
Microbiology, Paris, France) for the presumptive identification of
Candida species. The different-colored colonies were confirmed by
microscopy to be distinct, transferred to tubes containing Sabouraud
dextrose agar and maintained at 4[degrees]C until identification.
Phenotypic identification of the isolates was based on the germ tube
test, hypha/pseudohypha and chlamydoconidium formation, and carbohydrate
fermentation and assimilation OR germinative tubes, hyphae/pseudohyphae
and chlamyidoconidia formation, fermentation, and assimilation of
carbohydrates.

Anti-Candida IgA analysis in saliva

Saliva samples containing 5.0 mM phenylmethylsulfonyl fluoride and
0.002% sodium azide were stored at -20[degrees]C until antibody analysis
by ELISA. C. albicans ATCC 10231 grown in Sabouraud broth for 48 h at
37[degrees]C was used as the source of antigen. C. albicans yeast cells
killed with thimerosal (0.2 g/L) were harvested by centrifugation and
washed three times with 125 mM Tris-HCl (pH 6.8). The antigens were
extracted from cells by boiling in 125 mM Tris HCl (pH 6.8), 20 mM
2-mercaptoethanol, and 6 M urea for 5 minutes. The boiled product was
then harvested at 10,000 x g for 30 minutes at 4[degrees]C, and the
supernatant was dialyzed three times against 3 L distilled water and
then lyophilized.

Flat-bottomed 96-well plates (number 3590, Costar, Cambridge, USA)
were sensitized with 50 [micro]L of crude antigen solution (100
[micro]g/mL) dissolved in 0.1 M carbonate buffer (pH 9.6) and then
incubated for 2 h at 37[degrees]C and overnight at 4[degrees]C. The
wells were blocked with 0.5% gelatin (G) in phosphate-buffered saline
(PBS) for 1 h. Then, the plates were washed five times with 0.5% Tween
20 PBS (T-PBS) and incubated with 50 [micro]L of saliva sample diluted
1:8 in T-PBS plus 0.5% gelatin for 2 h at 37[degrees]C. After an
additional wash step with T-PBS, the wells were filled with
peroxidase-labeled goat anti-human immunoglobulin G, A, or M, and
incubated for 1 h at 37[degrees]C. Finally, 100 [micro]L/well of
o-phenylenediamine in 0.1 M citrate buffer (pH 5.5) was added at room
temperature. The reaction was stopped with 2.5 M [H.sub.2]S[O.sub.4] and
the absorbance was measured at 490 nm (3550--Bio-Rad Laboratories,
Hercules, USA). The results were expressed as values of optical density
(OD) obtained from the mean of two readings.

Exfoliative cytology

After oral mucosa specimen collection, the slides were immediately
fixed in a 1:1 solution of ether and alcohol and stained using the
Papanicolaou method. The slides were analyzed under microscopy by a
single researcher for the presence of yeast, which were counted in five
randomly selected fields. Sixteen graded reticles were evaluated in
every field, for a total of 80 reticles (200x). The number of cells with
and without Candida and the quantity of hyphae and/or blastoconidia was
counted.

No significant differences (p = 0.843) between the CFU/mL of yeast
species in saliva were observed between the EG and the CG. The EG
exhibited 1.159 [+ or -] 1.33 CFU/mL whereas the CG exhibited 1.228 [+
or -] 1.37 CFU/mL (values are expressed in [log.sub.10].

A higher incidence of non-albicans Candida was observed among
children in the EG (55.2%) compared with those in the CG (42.9%). The
species of Candida observed in the studied groups are presented in Table
1.

Anti-Candida IgA in the saliva samples was analyzed by ELISA. OD
values obtained for the groups were analyzed by ANOVA, and no
significant differences were observed between the groups (p = 0.16). The
mean OD values obtained were 0.024 [+ or -] 0.03 for the EG and 0.041 [+
or -] 0.05 for the CG.

To compare the IgA levels of the patients positive or negative for
yeasts in the saliva, the groups were subdivided into four groups:

a. EG1, patients who had orthodontic appliances and yeasts in their
saliva (n = 15);

b. EG2, patients who had orthodontic appliances but no yeasts in
their saliva (n = 15);

c. CG1, control patients with yeasts in their saliva (n = 16); and

d. CG2, control patients with no yeasts in their saliva (n = 14).

No statistically significant differences were observed when the IgA
counts in these groups were compared (p > 0.05; Table 2).

The exfoliative cytology results between the EG and CG children
were significantly different (p < 0.05). Group EG1 was similar to EG2
and different from CG1 and CG2 (p < 0.05; Figure 1).

Discussion

This study showed no differences in the presence of Candida in the
saliva of children in the EG and those in the CG. However, children in
the EG showed more yeasts adhering to their epithelial cells in the
exfoliative cytology examination. Hagg et al. (11) also observed that
candidal carriage did not significantly increase due to the insertion of
fixed orthodontic appliances using oral rinse or pooled plaque
techniques, but an increase was detected using the imprint culture
technique. According to these authors, the imprint technique is
sensitive to the localization of yeast growth and the oral rinse
technique is used to evaluate oral yeast carriage. (11) Similarly,
examination of exfoliative cytology is better than evaluation of saliva
to demonstrate adhering microorganisms. Our finding that the EG had
higher counts of adhering Candida suggests that the use of orthodontic
devices may cause stress or microtrauma in the oral mucosa that can
affect the process of microbial colonization and the adherence of yeasts
to epithelial cells. (8)

C. albicans was the species most frequently isolated from the oral
cavities of patients in both groups, followed by C. tropicalis. Among
the many factors that contribute to the higher prevalence of C. albicans
in the oral cavity are its excellent ability to adhere and the presence
of many cell receptors, which confer versatility and resistance to
removal by the fluids that bathe these surfaces. (8)

In this study, patients in the EG had a higher incidence of
non-albicans species in saliva as compared with patients in the CG.
Several studies have reported that predisposing factors such as mouth
breathing (12) and HIV-infection (13) may increase the variability of
Candida species in the oral cavity.

No correlation was observed between the level of anti-C. albicans
IgA in saliva and the presence of Candida or its adherence to epithelial
cells. The quantity of IgA was low in children of both the EG and the
CG. Salivary IgA generally increases with age because the secretory
immunological mechanism develops simultaneously with the humoral immune
system. (14)

Koga-Ito et al. (12) did not observe differences in the levels of
anti-C. albicans IgA in the saliva of treated and untreated children
with mouth-breathing syndrome and control children. However, Goncalves e
Silva et al. (15) observed that patients with clinical diagnoses of
vaginal candidiasis exhibited lower levels of anti-Candida IgA in the
saliva.

According to San Millan et al., (9) in patients with dental
prosthesis or removable orthodontic devices, salivary IgA reduces the
adherence of C. albicans to polystyrene. Although the present study did
not assess the adhesion of Candida to the surface of the orthodontic
device nor the role of IgA in this phenomenon, our results indicate that
anti-C. albicans IgA is not the most important factor to determine
Candida carrier status.

Conclusion

Orthodontic appliances may favor the adherence of Candida species
to epithelial cells but did not influence the presence of these yeasts
in saliva. The levels of anti-C. albicans IgA in saliva did not
correlate with the presence of Candida or its adherence to epithelial
cells.

Acknowledgements

We thank Barbara Maegi and Ana Paula Barbosa for their help in the
collection and analysis of the samples.